PROJECT SUMMARY Mast cells play pivotal roles in the development of allergic inflammation via signaling- dependent secretion of various pro-inflammatory compounds (e.g., histamine and cytokines) that are stored in secretory granules. However, how are distinct populations of secretory granules differentially regulated to release their specific contents (called degranulation) is poorly understood. Our long-term goal is to delineate signaling-triggered regulation of mast cell secretion, revealing unique components or pathways that are amendable to biological or small molecule interventions. The overall objective of this application is to distinguish the different membrane fusion apparatuses used by mast cells to release key mediators. Based on findings from cell-based secretion assays, localization studies and reconstitution, including preliminary data from our own lab, we propose in our central hypothesis that mast cells secrete histamine, serotonin and TNF? by exploiting distinct but overlapping combinations of SNARE/SM proteins, in a fashion that is regulated via Munc18 phosphorylation. To test this hypothesis, we will exploit a multifaceted strategy that includes genetics, biochemistry and quantitative proteomics to pursue the following two specific aims. Aim 1 will define the respective SNARE/SM fusion apparatuses for histamine, serotonin and TNF? release. Aim 2 will assess the regulation of mast cell exocytosis via Munc18 phosphorylation. We expect the study to set the stage for uncovering signal transducers that connect signaling cascades directly to the release of specific mediators.